Failure of neural tube closure is a complex genetic disorder, rarely if ever seen as a simple Mendelian trait. In the clinical population, NTDs are probably most often caused by partial inactivation and/or gain of function mutations of multiple genes, acting in conjunction with the gestational environment. Recognizing such changes as disease associated mutations rather than simple polymorphisms is a challenge for assessment of risk in individual families. Another challenge is determining the most appropriate means to reduce NTD occurrence for a particular couple. Recent studies from these three collaborating laboratories lead us to hypothesize that a systematic examination of gene network interactions in mouse models will elucidate patterns that are applicable to risk assessment and prevention of NTDs in humans. In a mouse line with a folic acid (FA) responsive NTD, we identified a point mutation in Lrp6, a co-receptor for Wnt canonical signaling, which implicates several important developmental genetic pathways likely to be encountered clinically. Using this Crooked tail (Cd) mouse, we identified a possible gene expression and metabolism "signature" in liver tissue and blood, predicting a genetic make-up that will respond to FA supplementation by preventing NTD. In addition, we used ENU mutagenesis screens to identify NTD associated mouse mutations that provide candidates for human testing and which can be tested for response to FA and other potential NTD preventive agents. We propose to pursue a multi-institution effort to characterize genetic susceptibility profiles in mice and identify genetic backgrounds that respond to prevention measures such as dietary supplementation. Experiments will use FA and inositol supplementation to test whether these agents prevent NTD in Lrp6 null and NTD-prone mice with defined ENU-generated mutations. Gene transcript arrays will examine expression in the neural tube during closure in these mutants using cluster analysis to compare patterns in the mutant vs. wildtype siblings. In addition, the genetic and metabolic signature identified in Cd non-neural tissues will be compared against signatures in other ENU-generated and naturally occurring NTD mouse mutants to determine the predictive power of this pattern. This project will build a framework for rational approaches to new treatments based on molecular pathways. To this end, the relationship between the biochemical pathway that is disrupted and the responsiveness or resistance to FA supplementation will be examined in several mouse lines. This will begin to reveal mechanisms by which FA, and inositol, acts to suppress NTD.